Individual recognition of female hamsters by males: Role of chemical cues and of the olfactory and vomeronasal systems

Physiology & Behavior, Vol. 33, pp. 95-104. Copyright©PergamonPress Ltd., 1984. Printedin the U.S.A.

0031-9384/84$3.00 + .00

Individual Recognition of Female Hamsters by Males: Role of Chemical Cues and of the Olfactory and Vomeronasal Systems R O B E R T E. J O H N S T O N A N D K U R T R A S M U S S E N

D e p a r t m e n t o f Psychology, Cornell University, Ithaca, N Y 14853 R e c e i v e d 23 N o v e m b e r JOHNSTON, R. E. AND K. RASMUSSEN. Individual recognition of female hamsters by males: Role of chemical cues and of the olfactory and vomeronasal systems. PHYSIOL BEHAV 33(1) 95--104, 1984.--SexuaUy satiated male hamsters preferred to investigate and to mount an anesthetized, estrous, novel female over a similarly presented female with which the male had become satiated (the Coolidge effect); likewise, such males preferred a novel female recently mated with another male over the familiar female but showed no preference between fresh and mated novel females. Thus the Coolidge effect is at least partly dependent on discrimination of a new female by chemical cues. Another experiment indicated that transfer of a male's own scent during mating is not involved in discrimination between familiar and novel females. Flank gland secretion of females were sufficient for individual discrimination by males, whereas head region scents and vaginal secretions were not sufficient. The presence of female's flank glands was not, however, necessary for such discrimination. Lesions of or removal of the vomeronasal organ did not disrupt the preferences of sexually satiated males for a novel female, but elimination of main olfactory system function by ZnSO4 treatment of the olfactory mucosa did abolish such preferences. Thus olfactory cues are sufficient for individual discrimination of novel females by sexually satiated male hamsters, and such recognition leads to increased sexual arousal. These processes are mediated by the main olfactory system but not the vomeronasal accessory-olfactory system. Chemical communication Sexual behavior Hamsters (Mesocricetus auratus) Learning Reproduction Accessory olfactory system

Olfaction Vomeronasal organ Pheromones Coolidge effect Individual recognition Mate recognition

W I T H I N the last ten years there has been an explosion of interest in the vomeronasal organ and accessory olfactory system, stimulated in large part by the discovery of separate, non-overlapping projections of the main olfactory and accessory bulb [25] and from the first report of an apparently unique function for this system in the copulatory behavior of male hamsters [22]. As experimental results have accumulated, preliminary attempts to characterize the unique functions of this system have been put forward. In an extensive review of vomeronasal organ anatomy and function, Wysocki [27] suggested that it might tend to be particularly important in reproductive functions at both the behavioral and neuroendocrine levels of analysis. More recently, Meredith [19] has suggested that the vomeronasal organ might be especially important in responding to specific chemical signals (pheromones) whose functions are not dependent on directly relevant learning experiences. In a complementary fashion he suggests that the main olfactory system might come to subserve some of the same functions through learning. In the course of this excitement over the vomeronasal organ and its functions, the main olfactory system has been somewhat neglected, and Johnston [13] has made a preliminary attempt at comparing the two systems and characterizing the differences in function between them. Expanding on Meredith's theme, Johnston suggested that the main olfac-

tory system may be particularly important in social communication when the functions subserved were necessarily dependent on learning and/or when the basis for such communication was discrimination between complex mixtures of chemicals. Discrimination of individuals is a type of communication that is probably based on both these processes, and thus ought to be an olfactory system function. One situation in which discrimination of individuals may be important is known as the Coolidge effect, in which a male is allowed to mate to satiety with one female and is then tested with either the same female or a new female. The effect that has been observed in some species (e.g., rats, hamsters, meadow voles and grasshopper mice) is that males show higher levels of sexual performance in the presence of a new female than in the presence of the familiar female (see [5] for review). The interpretation usually given or implied is that the effect occurs because the male can tell when a new female is present, although there is little proof available for this interpretation (see below). Nonetheless if this is the correct explanation for the effect in hamsters, the Coolidge effect situation would provide an excellent test of the hypothesis that discrimination is an olfactory system function, since ability to discriminate should be relfected in the degree of sexual arousal of a sexually satiated male. Previous research has clearly shown that in male hamsters both the vom-

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eronasal system and the olfactory system have important influences on copulatory performance [18, 20, 22, 26] but the role of these systems in mate recognition has not been investigated. Despite the substantial literature on the Coolidge effect, there has been little investigation of the mechanisms involved. One of the primary alternative explanations for recognition of a new female by males is that novel, unmated females may court the male more vigorously than the familiar female and thus elicit more sexual behavior from him. This alternative has been discounted in several studies, such as those on hamsters [3], by pairing the male with either the familiar female or a novel female that has mated with another male. These experiments still leave open the possibility, however, that .the female also recognizes a new mate and courts him more avidly. Indeed, Lisk and Baron [16] have recently shown that female hamsters do recognize a new male and are more interested in him than in a familiar male. A second level of question about behavioral mechanisms regards the sensory basis of individual discrimination. Among rodents the most likely channel would appear to be olfactory, and odors have been demonstrated to be sufficient for such recognition in rats [4]. F o r example, males that had previous mating experience with several females were allowed to mate to satiety with one female; they then preferred the odors of a novel female to those of their original partner. Other cues could be important, perhaps the most likely being auditory cues. In hamsters, for example, ultrasonic calling by both males and females is a prominent part of courtship activities [6,7]. In the first experiment in this paper we investigated the mechanisms underlying the Coolidge effect in hamsters to determine if the effect was at least in part due to discrimination of a new female and if chemical cues were sufficient for such discrimination. The basic paradigm involved presenting sexually satiated males with two anesthetized females and observing the degree of interest and sexual behavior they displayed to each. EXPERIMENT 1

Method All hamsters (Mesocricetus auratus) were laboratory stock derived from the Lakeview-Charles River randombred strain. Individuals were caged separately in solidbottom polycarbonate cages and maintained on a reversed light schedule of 14 hr bright (35.3 Ix) and 10 hr dim (0.014 Ix) illumination. Food and water were always available. In Experiment 1, 34 adult males were used as subjects and were randomly assigned to one of three groups (12 in Condition 1, 12 in Condition 2, and 10 in Condition 3). All males were sexually experienced, having spent three 20 min periods with three different receptive females; during these sessions all males mounted vigorously and repeatedly (this regime of sexual experience is the same throughout this paper). Additional males from the colony were used as needed for mating with some of the stimulus females. Forty females from the laboratory colony served as stimuli; most were used more than once, but not in the same experimental condition. Females were anesthetized with sodium pentobarbital (60 mg/kg) for use as stimulus animals. The testing procedure was as follows. The day before a test, subject males were habituated to the testing arena for l0 min. At the beginning of a test a male was allowed to interact with a naturally estrous female in an observation box until

satiety, which we defined as 10 min without an attempt to mount; the duration of such interactions was usually 50-60 min. After meeting this criterion males were returned to their home cages for 8 min and then introduced into a 61 ×91 ×30 cm testing arena. This arena was made of painted wood except for a front wall of glass to facilitate observation. During the 10-min test, males encountered two naturally estrous but anesthetized females placed 15 cm apart. These females were placed ventral side down on top of a 6.4× 12.7x 1.3 cm wooden block so that their elevation from the floor and position approximated that of lordosis; the females' rear ends extended over the edge of the block to facilitate investigation of the genital region by the males. The amount of time that males spent sniffing the body, sniffing or licking the genital region, and attempting to mount each of the females was recorded with an event recorder. Mounting was defined as crawling on, grasping and pelvic thrusting. These procedures were the same for all experiments in this paper, except where noted. In this first experiment there were three conditions that differed in the stimuli that were presented to the male. In Condition 1, a male encountered the same female he had just mated with (SAME F E M A L E ) and a female that was novel to him and that had not recently mated ( F R E S H F E M A L E ) . This first condition replicates the Coolidge effect paradigm, with the difference that the male is given a simultaneous preference for two anesthetized females instead of independent sequential tests with awake females. In the second condition the stimulus females were the SAME F E M A L E and a female that was novel to the male but had just copulated with a second male (MATED F E M A L E ) . The purpose of this condition was to determine whether the Coolidge effect was due to cues from novel females regardless of whether they had mated or not. In the third condtiion males encountered a M A T E D F E M A L E and a F R E S H F E M A L E , both of which were novel to the males. If individual identity is the most important factor influencing the behavior of sated males, they should be stimulated by both of these types of females and show no preference between them. On the other hand, if males tend to avoid recently mated females they should prefer the F R E S H F E M A L E .

Results and Discussion Males that had mated to satiety with one female (SAME F E M A L E ) directed more attention to a second, novel female ( F R E S H F E M A L E ) in the subsequent preference test (Condition 1, Fig. 1). Males spent more time sniffing the body, sniffing the genital region, licking the genital region, and attempting to mount the F R E S H F E M A L E (p<0.01 for all measures, Wilcoxan signed ranks text). The most striking differences were observed in the lick genitals category. Not one male licked the gential region of the SAME F E M A L E , whereas 11 of the 12 males licked the genital region of the F R E S H F E M A L E . These results parallel those obtained with the standard Coolidge effect paradigm [3],. suggesting that our method measures the same behavioral tendencies, namely a renewed sexual arousal caused by an encounter with a novel female. Because chemical cues are the predominant ones available to males in our experiment, and since the taste system is unlikely to be involved in such discriminations, the results suggest that males use odors to distinguish between individual females. Males in Condition 2 that were presented with a choice between S A M E F E M A L E and a novel female that had just

INDIVIDUAL RECOGNITION OF FEMALE HAMSTERS [-1 Some female [ ] Fresh female •

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differences. More time was spent sniffing the genitals (p <0.01, Mann-Whitney U) and licking the genitals (p <0.01) of the F R E S H female than of the M A T E D female; differences between Sniff Body and Mount categories were not significant. Thus, under some circumstances odors produced during copulation may have an effect on the subsequent behavior of males. The results of Condition 3, however, indicate that the importance of such an effect is slight. Bunnell et al. [3] reported that satiated males were more sexually activated by F R E S H females than by M A T E D females, but in their testing situation differences in the behavior of the females may have been the most important variable, since the receptivity of estrous, unmated females is much greater than the receptivity of estrous, once-mated females [16].

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An alternative explanation for some of the results found in Experiment 1, in particular those of Condition 2 in which males preferred M A T E D F E M A L E S over S A M E F E M A L E S , would be that males were making a discrimination between females that had some of the male's own odor on her versus the odors of another male. This would mean that the effect would still be due to discrimination of individual differences, but that the odors of males were the important ones. If true, it would dramatically alter hypotheses about the functional significance of this effect. We evaluated this possibility by testing satiated males with two novel females, one of which had the males' own odors transferred onto her.

MOUNT

FIG. 1. Mean number of seconds (-+SEM) that sexually satiated

males spent directing investigative and sexual activities at one of two estrous but anesthetized stimulus females. The females were one of the following: SAME---female the male had just copulated with: FRESH--a novel, unmated female; MATED---a novel female that had just mated with another male. (**,o<0.02, ***p<0.0I, onetailed Wilcoxon signed ranks test).

mated with another male ( M A T E D F E M A L E ) showed greater levels of interest in the novel, M A T E D F E M A L E (Fig. 1). Once again, the differences were significant for all of the behavioral categories we measured: Sniff Body (p <0.01), Sniff Genitals (p<0.01), Lick Genitals (p<0.02), and Mount (p<0.01). These results indicate that the renewed sexual interest shown by males is primarily due to their discrimination of a new individual female. They rule out the possibility that males become uninterested in females because of odors produced during copulation. When presented simultaneously with two novel anesthetized females, one F R E S H and the other MATED, males showed no significant preferences as assessed by the measures of their behavior that we recorded (Condition 3, Fig. 1). The results of this condition strongly support the interpretation stated above that the important information that results in arousal of satiated males is that indicating a different individual. In the third condition, odors produced during copulation and present on the mated females had no measurable effect on the males' preferences. However, if we compare the behavior of the males in Condition 1 toward F R E S H F E M A L E S and the behavior of the males in Condition 2 toward M A T E D F E M A L E S , we do see some significant

Method

Twelve sexually experienced male hamsters 4-6 months of age were used as subjects. Thirty-six females were used as stimulus animals. The procedure was similar to that used in Experiment 1. Three females were required to test each male. A male was allowed to mate to satiety with the first female. During the eight-minute break before the test began the male's odors were transferred to one of the stimulus females ( F R E S H , SCENTED) by (1) rubbing a glass rod over the male's flank gland for 30 sec and then rubbing this on the flanks of the previously anesthetized female for 30 sec, (2) rubbing the glass rod over the head of male for 30 sec and then rubbing the female for 30 sec, and (3) picking up the male and rubbing him against the female, particularly on his ventral surface and genital region for 60-75 sec. The F R E S H , S C E N T E D F E M A L E and the F R E S H F E M A L E were then placed in the testing arena as described in Experiment 1, the male was introduced, and his behavior was recorded during a 10 min trial. Results and Discussion

Males were quite interested in both the F R E S H , S C E N T E D female and the F R E S H F E M A L E . The mean total time ( - S E M ) spent sniffing, licking and mounting the F R E S H , S C E N T E D F E M A L E S was 146.9±9.8 sec, while the mean for the F R E S H F E M A L E was 143.2_+13.8 see (Fig. 2). Neither this difference nor any of those for the individual categories of behavior were significant. The mean ( ± S E M ) number of seconds involved in the individual categories o f behavior for the F R E S H F E M A L E and then the F R E S H , S C E N T E D F E M A L E were as follows: Sniff Body, 63.6±8.0 sec vs. 79.6± 13.3 sec; Sniff Genitals,

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JOHNSTON AND RASMUSSEN 160

EXPERIMENT 3

T In this experiment we investigated the possible sources of scent that allow individual discrimination and arousal of satiated males. There are four discrete sources of scent that have been clearly shown to have communicative significance in hamsters (see [l 1,14] for reviews). These include vaginal secretions and the secretions of the sexually dimorphic flank, ear, and Harderian glands. Since ear and Harderian secretions are mixed together when an individual grooms, we decided to test these together as scents from the head region and compare them to scents from the flank region and vaginal secretions.

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FIG. 2. Mean number of seconds (-+SEM) that sexually satiated males spent sniff'mg,licking and mounting one of two novel females, one of which (FRESH, SCENTED) had been scented with the male's own odor.

11.5---2.8 vs. 7.6_+1.3 sec; Lick Genitals, 31.1-+6.2 vs. 36.1-+ 13.4 sec; Mount, 38.9-+7.6 vs. 32.5---5.3 sec. Transfer of scent from the test male to a novel female did not render that female less attractive or less arousing than a novel, unscented female. These results reinforce the hypothesis that the primary factor which stimulates satiated males is the recognition of a new individual female. It could be argued that our methods of transferring odor were not effective, but this seems unlikely. Other hamsters tested with the glass rods we used spent more time s ~ g ones that had been rubbed on a male than clean glass rods, indicating that we were successful in obtaining scent. In other experiments similar rubbing techniques have been quite successful in providing scent on glass for stimulus materials [15]. It could also be argued that we did not transfer the appropriate odors to the female, but this too seems unlikely, since we rubbed most of the males' body surface in one way or another, including considerable attention to the ventral surface and genital region, which are the areas most likely to contact the female during copulation. We did not specifically transfer ejaculate to the stimulus female, although some of this scent was probably transferred when we rubbed the male's genital region against the female. It would be interesting to determine if males could distinguish other males by the odor of the ejaculate and if such discrimination would influence their sexual behavior. We conclude from this experiment that it is unlikely that the Coolidge-type effect we observed was due to the discrimination of males' odors on the females. It is much more probable that the effect is due to discrimination between individual females.

Thirty-six sexually experienced males 6--12 months of age were divided into three groups of twelve for use as subjects. Forty females, 2-6 months old, were used as sexual partners or as sources of scent. Sexually satiated males (as defined in Experiment 1) were tested in a manner similar to that described in the previous experiments, except that instead of encountering two anesthetized females in the test chamber they encountered two 7.6x 12.7 cm glass plates on the floor of the arena 15 cm apart. One of these plates was scented with odors from the female that the male had just mated with (SAME FEMALE), while the other was scented with odors of a novel, unmated female (FRESH FEMALE). Separate groups of twelve males were tested with odors from the head region, flank region, or vaginal secretions. Trials lasted for 5 min, during which the amount of time that the males spent sniffing and/or licking at each of the glass plates was measured. The stimulus plates were prepared in the following manner. The plates were first cleaned by washing them with sodium lauryl sulfate, rinsing with water, rinsing with ethanol, rinsing with water, and air drying. To obtain flank gland and head region scents, the glass plates were rubbed continuously on these areas for 30 sec. When rubbing the head region, we were careful to include the ears, eyes, and corners of the mouth. Vaginal secretions were obtained by extruding vaginal secretions onto a stainless steel spatula and transferring them to the glass plate. The experimentor wore surgical gloves when handling the stimulus plates in order to reduce the amount of extraneous human scent deposited on them. Results and Discussion Sexually satiated males were interested in scents from all three body regions of females and spent considerable time sniffing and/or licking at them (Fig. 3). Males in the flank scent condition were more interested in the flank scent from FRESH F E M A L E S than that from SAME F E M A L E S (p<0.005, one-tailed t-test), but males in the other two scent conditions did not demonstrate significant preferences. These results thus demonstrate that odors from the flank gland region are sufficient for individual discrimination of female hamsters and suggest that flank gland odors may be particulary important for individual discrimination and the sexual arousal in satiated males that is dependent on such discrimination. The lack of preference for odors of the head region or vaginal secretions suggests these scents are relatively unimportant for individual discrimination, at least as we measured it.

INDIVIDUAL RECOGNITION OF FEMALE HAMSTERS ["'7 Same female

30

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99 tal region, 3.2_+1.0 at the S A M E F E M A L E vs. 15.2___1.5 at the F R E S H F E M A L E (0<0.005); (3) licking genitals, 1.9-+1.1 at S A M E F E M A L E vs. 47.5_+12.4 at F R E S H F E M A L E (0<0.005); (4) mounting, 1.0_+0.7 at S A M E F E M A L E vs. 32.1___10.0 at F R E S H F E M A L E (,o<0.005). The lack of flank glands did not hinder the males' ability to discriminate the familiar from the novel female, nor did it result in a lack of sexual arousal toward the novel female. Thus, although the flank gland secretions are sufficient for individual discrimination and appear to be more important than scents from the head region or vaginal secretions, they are not necessary. EXPERIMENT 5

In this experiment, we investigated the role of the vomeronasal organ and the accessory olfactory system in the sexual arousal of sated males caused by the recognition of a new partner. He~l

Flank

Vag~n litml~c.

SOURCE OF SCENT

FIG. 3. Mean number of seconds (+SEM) that sexually satiated males sniffed vaginal, head or flank secretions from females they had just mated with (SAME) or'novel female (FRESH). (*p<0.005, one-tailed t-test).

EXPERIMENT 4

In this experiment we investigated whether the flank gland of females is necessary for discrimination of individuals by males. Method

Twelve sexually experienced males, seven months old, were used as subjects. Sixteen female hamsters served as stimulus animals. All of these females had their flank glands surgically removed 3--6 weeks prior to the experiment. Surgery was performed under sodium pentobarbital (60 mg/kg) anesthesia, and involved simple removal of two small pieces of skin that included the flank glands. Some of these females were used to test two males, but each pair of females used was unique. As usual, males were allowed to mate with one female until the criterion of satiety was met, 10 minutes without a mount. The male was returned to his home cage and 8 min later placed in the testing arena, in which he encountered two anesthetized females. These females were the S A M E F E M A L E that he had just copulated with and a female that was not familiar to him and that had not recently copulated ( F R E S H F E M A L E ) . This procedure was exactally the same as that used in Experiment 1, except that both stimulus females lacked their flank glands. Results and Discussion

Sexually satiated males demonstrated strong preferences for F R E S H F E M A L E S over S A M E F E M A L E S , despite the fact that both females lacked flank glands. They spent significantly longer times sniffing the body and genital region, licking the genital region, and mounting the novel female, just as males in Experiment 1 did when tested with intact females. The mean times (_+SEM) spent in various activities were as follows: (1) Sniffing the body, 20.2_-.4.7 sec at the S A M E F E M A L E vs. 86.3-+16.1 sec at the F R E S H F E M A L E (0<0.005, Wilcoxon signed ranks test); (2) sniffing the geni-

Method

Twenty-three sexually experienced male hamsters were used as subjects, 14 in the lesioned group and 9 in the shamlesioned control group. Vomeronasal lesions were made following the procedure of Johns et al. [10]. Males were anesthetized with sodium pentobarbital (60 mg/kg). An electrode was made of copper wire that was flattened to a knife edge, 0.5 cm long, on one side; it was insulated with epoxy except for the fine edge, which was bare. The electrode was inserted into each nostril, one at a time, and positioned in the groove on the ventral surface of the nasal cavity; a second ground electrode was attached to the cheek. Using a Grass LM-3 radio frequency lesion maker, a potential of 5 volts was applied for I0 sec. In the sham lesion group, the electrode was inserted so that it contacted the dorsal surface of the nasal cavity, and the same voltage was applied. Thus a lesion was produced in the non-olfactory nasal epithelium. Two days after the lesions were produced the males were tested, first by allowing them to mate to satiety and then giving them a choice between the S A M E F E M A L E and a F R E S H F E M A L E , as described in detail in Experiment 1. Males were later sacrificed, and their nasal cavities prepared for histology following the methods of Winans and Powers [26]: after decalcification the snouts were embedded in paraffin, sectioned at 40/x and stained with either cresyl violet or hematoxylin and eosin. The histology of the vomeronasal organs for 8 lesioned and 3 sham control animals was analyzed quantitatively. Nine sections at equal intervals along each animal's vomeronasal organs were examined, starting with the first section with a recognizable organ. The extent of damage was determined by projecting the slide onto graph paper and calculating the percentage of sensory epithelium that was destroyed. The mean of the two sides was used as a measure of epithelium damage at t h a t level. These data were then used to obtain group averages. All sections containing olfactory epithelium were also examined for evidence of tissue damage. Results

Males with vomeronasal lesions and sham lesions continued to discriminate between the S A M E F E M A L E S and the F R E S H F E M A L E S and to direct more of their investigation and sexual interest at the F R E S H F E M A L E S (Fig. 4). The total time spent in such activities by the lesioned males was 20.1-+3.5 see at the S A M E F E M A L E vs. 96.8_+13.4 see at

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epithelium but no evidence of damage to the olfactory epithelium. In all males 100% of the epithelium in the first section of the vomeronasal organ was destroyed and in 7 of 8 males the first four sample sections were 100% destroyed. Posterior to the fourth sample section damage varied from 0-100%; 2 males showed complete destruction of the vomeronasal epithelium. Thus the anterior lh-1/2 of the vomeronasal organ was destroyed in all but one male, whereas the posterior part appeared normal in the majority of males. Chemicals gain access to the interior of the organ via the pore in the anterior end, and presumably the lesion prevented stimulus access from occurring. Similar interpretations of this type of lesion have been made by other authors [10]. We were concerned about this lack of complete destruction of the organ, however, so we tested another group of males in which the vomeronasal organs were surgically removed. Vomeronasal organs were surgically removed from seven sexually experienced males (8-10 months old) using procedures developed by Wysocki [28]. After anesthetization (sodium pentobarbital, 60 mg/kg) males were placed on their backs, the mouth was held open, the ventral surface of the nasal cavity exposed, and the vomeronasal organ and part of its capsule removed with a forceps. Three to four months later males were paired with an estrous female and allowed to mate; six of the seven did so. One week later they were subjected to the standard testing paradigm, first mating to satiety with one female and then being exposed to two anesthetized females (SAME F E M A L E and FRESH F E M A L E ) for ten rain. Six of the seven males mated vigorously with the awake female but the seventh did not mate (not the same one that failed to mate previously). Of the six that did mate, all six spent more time investigating and mounting the F R E S H F E M A L E than the S A M E F E M A L E , 76.5___21.6 sec vs. 13.2_+2.5 sec (O<0.02, Wilcoxon signed ranks test). The measures of three of the components that made up this total were also significantly different: 10.6 vs. 53.7 sec sniffing the body (O<0.02), 1.2 vs. 7.2 sec sniffing genitals (O<0.03), and 1.0 vs. 15.1 sec licking genitals (o<0.03). Only two of these males mounted the anesthetized females. Histological analysis showed that in 5 animals the vomeronasal organs were entirely missing and that there was no lasting damage to the olfactory epithelium. Thus in these animals there is little doubt that vomeronasal function was completely eliminated, and yet they too were more interested in the F R E S H F E M A L E than the S A M E F E M A L E . In two animals there were small, relatively normal looking pieces of the anterior organ. Since most of the organ was missing it was not possible to accurately estimate the percentage of tissue remaining, but roughly 1/4--1/3 of the organ remained in these two animals. Since the vomeronasal nerves pass caudally along the organ it is likely that these were removed with the posterior part of the organ. The behavior of these two animals was not discernibly different from those with complete removals. The results of these two experiments, in which we used two different methods of eliminating vomeronasal organ function, indicate that sexually satiated males that lack vomeronasal organ function demonstrate a greater degree of sexual interest and arousal toward a novel female than toward one they have recently mated with. Although some experimental males may have had some vomeronasal function remaining, the histology indicated that 2 males with vomeronasal lesions and 5 with vomeronasal removals had either complete lack of the organ or of its epithelium. The behavior of such males was not discernibly different from those with

--I VNOSHAM

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MOUNT

FIG. 4. Mean number of seconds (±SEM) that males with vomeronasal organ or sham lesions spent snitTmg, licking and mounting the SAME or a FRESH female. (*p<0.05, ***p<0.01 one-tail (Wilcoxon signed ranks test).

the F R E S H F E M A L E S (p<0.01, Wilcoxon signed ranks test). The sham lesioned males behaved similarly, spending a mean total time of 30.1--+ 10.9 sec with the SAME F E M A L E versus 102.4___24.6 sec with the F R E S H F E M A L E (o<0.05). As shown in Fig. 4 all of the individual measures of behavior patterns demonstrate a significant (or near significant) preference for t h e F R E S H F E M A L E S . Males with vomeronasal organ lesions sniffed and licked the genital region of the F R E S H F E M A L E more than that of the SAME F E M A L E , and, most importantly, they also mounted the F R E S H F E M A L E significantly more often. The sham-lesioned males showed similar results and the levels of behavior shown by the lesioned and control males were not statistically different. In the control group one male spent more time mounting the SAME F E M A L E , a big factor in the lack of significance of the overall trend of preferring to mount the F R E S H FEMALE. It is interesting to note that all 14 males with vomeronasal lesions did mate with awake females, a result that differs f r o m some previous reports in which the vomeronasal nerves were cut [22,26], but is in agreement with others [18, 20, 23]. During the test with two anesthetized females 8 of 14 lesioned males mounted at least one of the females while 6 of the 9 sham lesioned males did so. In Experiment 1, 9 of 12 intact males mounted one of t h e anesthetized stimulus females. Thus although all of the lesioned males did not mount the anesthetized females, this performance was not significantly different from sham lesioned or intact males, and other indices of sexual interest were high. F o r example, all 14 lesioned males sniffed the genital region of the F R E S H F E M A L E but only 4 sniffed the SAME F E M A L E ; 10 lesioned males licked the genital region of the F R E S H F E M A L E S , whereas only one licked the S A M E F E M A L E . All eight of the lesioned males that were histologically examined showed substantial destruction of the vomeronasal

INDIVIDUAL RECOGNITION OF FEMALE HAMSTERS partial lesions or removals, further suggesting that animals with partial damage may have had no vomeronasal function. On the other hand, the histology showed that the removal technique was more successful than the lesion technique in destroying vomeronasal function. This does correlate with a difference in the behavior of the two types of experimental males: satiated males with vomeronasal removals did not mount the anesthetized F R E S H F E M A L E as consistently as the satiated, vomeronasal lesioned males did. Thus it is possible that some of the sexual arousal shown by some of the vomernosal-lesioned males may have been due to paritial vomeronasal function. EXPERIMENT 6

101

60

40

ZnSO40LF. LESION 20

¢n 0

60

~.~

In this experiment we investigated whether the main olfactory system mediated the recognition of new females by sexually satiated males and the consequent increase in sexual arousal following such recognition.

40

Method

20

Seventeen sexually experienced males, 7-11 months of age, were used as subjects. Ten of these males had their olfactory mucosa destroyed by treatment with ZnSO4 and seven were saline-treated controls. Irrigation of the nasal cavities with ZnSO4 or saline was accomplished by procedures similar to those described by Powers and Winans [21]. Males were anesthetized with ether, laid on their backs and suspended head down; 0.85 cc of 0.5% saline or of freshly prepared ZnSO4 in saline was introduced into the rear of the nasal cavity and was allowed to drain out the nares. Clearance of these solutions was aided by gentle aspiration. Males were kept in a head-down position until they were almost fully recovered from the anesthesia. All animals were premedicated with atropine sulfate (0.0005 mg) to help control nasal congestion. Two days after the experimental or control treatments males were tested in the standard paradigm, first mating to satiety and then being tested with two anesthetized females, the S A M E F E M A L E and the F R E S H F E M A L E . The day after testing 7 of the 10 experimental males and 4 of the 7 controls were sacrificed for histological verification of the experimental treatments. Due to human error the histology from the experimental males was not complete enough for quantitative analysis; histology from these males was examined qualitatively and another seven males were treated with ZnSO4 and sacrificed for quantitative analysis. Three sections from each male, representing the middle of each third of the extent of olfactory epithelium, were examined; estimates of the damage to each half of the sensory epithelium were made using a ten-point scale (i.e., 0-10% normal epithelium, 10-20% etc.); two halves were averaged to arrive at a figure for each section. To assess possible damage to the vomeronasal organ we examined all sections containing vomeronasal sensory epithelium; since we found no damage in any of these sections a quantitative analysis was not necessary. Results

All males treated with ZnSO4 mated vigorously with their inital partners, but once they were sexually satiated they showed little interest in either of the anesthetized stimulus females. Sexually satiated, saline-treated control males demonstrated a clear preference for the F R E S H F E M A L E S

[---1 Same female Fresh female

SNIFF BODY

SALINECONTROLS

SNIFF GENITALS

LICK GENITALS

MOUNT

FIG. 5. Mean number of seconds (_+SEM) that males with ZnSO4 or saline treatment of the nasal mucosa spent sniffing, licking and mounting the SAME female or a FRESH female (*p<0.05, **p<0.02, Wilcoxon signed ranks test).

(Fig. 5). All of the control males sniffed and licked the genitals of the F R E S H F E M A L E S , whereas only one of them sniffed and licked the S A M E F E M A L E S . Five of the seven controls mounted the F R E S H F E M A L E ; 2 of the 7 mounted the S A M E F E M A L E . In contrast, most of the ZnSO,treated males showed no interest in investigating the genital region or in mounting: The median levels of behavior for Sniff Genitals, Lick Genitals, and Mount were 0.0 for both stimulus females. Eight of the ten ZnSO4-treated males demonstrated little or no sexual interest; one sniffed the genital region for 1 sec and one spent 1 sec mounting. Two males, however, spent a small to moderate amount of time sniffing and/or licking the genitals of the anesthetized females; these two spent 24 and 30 sec mounting the F R E S H F E M A L E and 2 and 6 sec mounting the S A M E F E M A L E . Quantitative histological examination of additional males revealed that destruction of the epithelium was quite thorough in most animals, the range in the mean amount of normal epithelium remaining was from 5% for several animals to 58% for one animal. Discounting the one animal with little damage, the mean percentage of normal epithelium was 9.4%, quite similar to that reported by Winans and Powers [26]. The histology of the experimental males qualitatively indicated the same large degree of destruction. Many of the sensory turbinates in these sections were folded over, making quantitative analysis difficult. Qualitatively, all but one of the 8 original experimental males that we examined histologically showed large scale destruction of the olfactory epithelium. The one with little damage was one of the two males that showed high levels of interest in the anesthetized females. The epithelium of other such male was not available for examination. Examination of the vomeronasal organs revealed no damage to the sensory epithelium.

102

JOHNSTON AND RASMUSSEN

These results thus suggest that discrimination by sexually satiated males of individual females, and the sexual arousal that depends on the discrimination of a novel female, depends on the main olfactory system. If this interpretation is correct we would expect that ZnSO4-treated males in the standard Coolidge effect paradigm would also be less sexually aroused than control males, since at least part of a male's reinvigoration in this context should be due to recognition of a novel female. In a subsidiary experiment we found this to be true. Fifteen sexually experienced males were used as subjects, 8 ZnSO4-treated experimentals and 7 salinetreated controls. They were allowed to mate to satiety, were returned to their home cage for 8 min, and then were exposed to a novel, naturally estrous female for 10 min. As seen in Table 1, males with olfactory system impairments showed significantly lower levels of licking the genital region of females, sniffing the female's genitals and bodies, mounting with correct orientation, intromitting and grooming their own genitals than saline-treated control males did. All males in both groups did engage in some sexual behavior, indicating that olfactory impairment did not totally eliminate the ability of a satiated male to become rearoused by an actively courting female. Nonetheless the sexually satiated ZnSO4treated males showed significantly less sexual behavior than control males, again suggesting that deficits in olfactory system function lead to a greatly diminished response to novel females. GENERAL DISCUSSION

The present experiments demonstrate that chemical cues from a new female are sufficient to reawaken the sexual interest of a male that is sexually satiated. Although visual cues were available to males in our standard test situation, sexual arousal was eliminated by incapacitating the olfactory system with ZnSO4, thus suggesting that chemical cues are necessary and that visual cues alone are not sufficient. Furthermore, our experiments provide a strong basis for the interpretation that the reawakening was due to recognition of a new individual rather than discrimination of a mated from an unmated female or of a female scented or unscented by the male in question. A similar interpretation could be placed on the results of previous olfactory preference studies with rats, in which it was shown that sexually satiated males preferred the odors of novel over familiar females [4]. Since in these experiments the odor stimuli were collected before mating occurred there was no question of transfer of male odors to complicate the analysis. In Experiment 3, sexually satiated male hamsters investigated the flank gland scent of a novel female more than the flank-gland scent of the familiar female, whereas a similar preference was not shown for head region odors or vaginal secretions of the novel female. These results suggest that the flank gland is a particularly important source of individually discriminable and therefore sexually arousing odors. The effects of removal of females' flank glands also implicate this scent as being involved in sexual arousal [12,13]. The current results indicate that the olfactory system mediates discrimination of individuals whereas the vomeronasal system dos not. We know of no other experiment that compares the two systems on this dimension, but several other reports fit with the more general hypothesis that the olfactory system, but not the vomeronasal system, may be important in social discriminations. Male hamsters prefer anesthetized conspecific females over heterospecific

TABLE 1 MEDIAN NUMBER OF BEHAVIORS SHOWN BY ZnSO4 AND SALINE-TREATED SATIATED MALES DURING A 10-MIN ENCOUNTER WITH AN AWAKE, NOVEL, ESTROUS FEMALE

Males Behaviors Sniff Body Sniff Head Sniff Genitals Lick Genitals Correct mounts Incorrect mounts Intromissions Own body groom Own genital groom

Saline Treated 14.0 5.0 11.0 17.0 13.0 2.0 13.0 12.0 19.0

ZnSO4 Treated $ * § e t *

7.5 5.5 3.5 1.0 8.0 1.5 8.0 11.0 15.5

*p=0.047, tp=0.02, $p=0.014, §p<0.001, Mann-Whitney U.

females. This preference is not dependent on the vomeronasal system, but is eliminated when the entire olfactory bulb is removed, thus eliminating the input from both systems and implicating a crucial role for the olfactory system [20]. The preference of male hamsters for the odors of females over the odors of males may also be dependent on the olfactory system, since lesions of the mediodorsal nucleus of the thalamus eliminate such preferences. This nucleus receives its input from the olfactory bulb via the olfactory cortex but does not receive input from the vomeronasal system [24]. In this case, however, the results could be explained by a difference in the responsiveness of the two systems to volatile chemicals, and thus may not reflect a unique role for the main olfactory system in sexual discrimination. In our experiments on individual discrimination and Murphy's [20] experiments on species preferences contact with the stimulus animals was possible, so the case for these functions being specifc to the olfactory system is much stronger. In contrast, it has been suggested that the vomeronasal system is important for "gender recognition" in mice [28]. The situation investigated was the tendency of socially experienced male mice to produce more ultrasonic calls in the presence of females or their odors than in the presence of males or their odors. Differential responses to urine odors were eliminated in males whose vomeronasal organs were removed before they had any social experience. Vomeronasal organ removal had little effect on males with social experience prior to the surgery, or on males exposed to the females themselves. Thus, although the vomeronasal organ may have been involved in the initial learning of chemical cues that allowed discrimination between males and females, the organ did not seem to be necessary for this process in socially experienced males or in naive males that had access to more than just urine cues. However, gender discrimination may be a very different process than individual or species discrimination in that it could be based on detection of as little as one or two sex-specific signals. Individual discrimination presumably involves a much more difficult discrimination between patterns of odorani concentrations, and it is this type of discrimination that we suggest is a unique function of the olfactory system.

INDIVIDUAL RECOGNITION OF FEMALE HAMSTERS In common with previous studies we found that damage to the olfactory epithelium by threatment with ZnSO4 did not seem to disrupt mating behavior of male hamsters when they were paired with awake, behaving females [17,21]. It could be argued that the results we observed with ZnSO4-treated, sexually satiated males were not due to their inability to recognize a new female but rather due to a deficit observed when such males are paired with anesthetized females, perhaps due to the additive effects of a sub-optimal stimulus animal and lower general arousal level due to the sensory deficit. However, in the pilot study reported above we found that ZnSO4-treated males that were sexually satiated and then exposed to a novel, estrous behaving female also demonstrated lower levels of sexual arousal than salinetreated control males did. Furthermore, in another experiment we observed nine ZnSO4-treated males that were just exposed to anesthetized females for 10 min. They spent a mean of 111.2 sec mounting the females and another 69.3 sec sniffing the body and sniffing and licking the genital region. Thus males with olfactory system lesions will readily mount anesthetized females if they have not been sexually satiated beforehand. We conclude males lacking a functional olfactory system cannot recognize the presence of a novel female, and therefore are not sexually rearoused by them. An interesting result observed in these experiments was the relatively high level of sexual activity observed in males that had either vomeronasal organ lesions or vomeronasal organ removals. All males with damage to the vomeronasal organ mated with naturally receptive females, although two males in the organ removal group failed to mate on one of the two mating tests. In a preliminary but more quantitative study [13] we confirmed the finding that control males and males with vomeronasal organ removal were not different in copulatory performance. None of our males would have fallen into the severe deficit category as originally defined by Winans and Powers [26]. In the Winans and Powers experiment 44% of all males with vomeronasal nerve cuts displayed such severe deficits. In a later study from the same lab [22], behavioral deficits were defined much less restrictively and yet the percentage of males showing such deficits was lower (25%) than in the original study. This later study seems similar to one reported by Murphy [20], in which 2 of 10 males with nerve cuts failed to mate. All of these deficits are more severe than the deficits we observed. On the other hand Meredith [18] reported no significant mating deficits after similar vomeronasal nerve cuts. Although it seems clear that both the olfactory and vomeronasal systems are involved in sexual arousal of male hamsters, it is not easy to reconcile all of these different results. The procedure of cutting the vomeronasal nerves necessarily involves damage and trauma to the olfactory bulb, so that severe behavioral deficits could be due to the combination of lack of vomeronasal input and reduced olfactory system input. Some suggestive evidence for this in-

103 terpretation comes from Winans' and Powers' [26] quantitative analysis of the lesion damage, in which they found some suggestions that the extent of damage to the main olfactory bulb was correlated with the extent of behavioral defict. Differences in the degree of behavioral deficits between different studies using this technique could in part be due to differences in the degree of olfactory bulb trauma. Nerve cut studies that minimize truama to the bulb and studies that incapacitate the vomeronasal organ itself may demonstrate the smallest deficits in sexual behavior. Another relevant difference between some of the experiments is the use of naturally cycling, estrous females as opposed to ovariectomized, hormone-treated stimulus animals. Naturally cycling estrous females display higher levels of sexual motivation than induced-estrous females and thus may elicit a greater response from males [1]. Winans and Powers [22,26] used hormone-treated females, whereas Meredith [18], Murphy [20], and the current experiments used naturally cycling females. It was shown some time ago that rodents could distinguish between the odors of different individuals [2], but it is only more recently that demonstrations of how they might actually use odors for individual recognition have been elaborated (see [9] for review). Although the specific paradigm used in the present experiments is necessarily constrained, it is likely that similar discriminations of individual sexual partners do take place and effect the mating success of individuals in natural populations. At least among some groups of mammals the capacity of sexually satiated males to become sexually aroused by a new female may be indicative of a polygynous or promiscuous mating system (see [5] for evaluation of this idea). The one counter example to this hypothesis among the rodents that Dewsbury discusses is the Northern grasshopper mouse, Onychomys leucogaster, a species that is supposed to be monogamous but that shows the Coolidge effect. The evidence for monogamy is weak, however, and the closely related species O. torridus is clearly not monogamous [8]. In hamsters, recent experiments suggest that sexually satiated females may show a similar type of Coolidge effect, mating for longer durations with a novel as opposed to a familiar male [ 17]. We would predict that where such recognition is based on chemical cues the olfactory system should mediate such recognition and the consequent sexual arousal.

ACKNOWLEDGEMENTS This research was made possible by NSF grants Nos. BNS7822623 and BNS-8210277/R to R. E. Johnston. The authors are grateful to Ann Meyer for help in developing the VNO-lesion technique, Chuck Wysocki for teaching us the VNO-removal technique, and to Mary Ascenzi, Alice Yih, and Becky Roberts for technical assistance.

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4. Cart, W. J., L. Krames and D. J. Costanzo. Previous sexual experience and olfactory preference for novel versus original sex partners in rats. J Comp Physiol Psycho l 71:216--222, 1970. 5. Dewsbury, D. A. Effects of novelty on copulatory behavior: The Coolidge Effect and related phenomena. Psychol Bull 89: 464--482, 1981.

104 6. Floody, O. R. and D. W. Pfaff. Communication among hamsters by high-frequency acoustic signals. I. Physical characteristics of hamster calls. J Comp Physiol Psychol 91: 794-806, 1977. 7. Floody, O. R., D. W. Pfaff and C. D. Lewis. Communication among hamsters by high-frequency acoustic signals, II. Determinants of calling by females and males. J Comp Physiol Psychol 91: 807-819, 1977. 8. Frank, D. Social organization of the southern grasshopper mouse, Onychomys torridus. Paper presented at the meeting of the Northeast Animal Behavior Society, Kingston, Ontario, 1982. 9. Halpin, Z. T. Individual odors and individual recognition: review and commentary. Biol Behav 5: 233-248, 1980. 10. Johns, M. A., H. H. Feder, B. R. Komisaruk and A. D. Mayer. Urine-induced reflex ovulation in anovulatory rats may be a vomeronasal effect. Nature 272: 446-448, 1978. 11. Johnston, R. E. Sex pheromones in golden hamsters. In: Chemical Signals in Vertebrates, edited by D. Mueller-Schwarze and M. M. Mozell. New York: Plenum Press, 1977, pp. 225-249. 12. Johnston, R. E. Chemical signals and reproductive behavior. In: Pheromones and Reproduction in Mammals, edited by J. G. Vandenbergh. New York: Academic Press, 1983, pp. 3-37. 13. Johnston, R. E. Olfactory and vomeronasal mechanisms of communication. In: Olfactory and Gustatory Influences on the Central Nervous System, edited by D. W. Pfaff. New York: Rockefeller University Press, 1984, in press. 14. Johnston, E. E. Communication. In: Hamster: Reproduction and Behavior, edited by H. I. Siegel. New York: Plenum Press, 1984, in press. 15. Johnston, R. E. and T. Schmidt. Responses of hamsters to scent marks of different ages. Behav Neural Biol 26: 64-75, 1979. 16. Lisk, R. D. and G. Baron. Female regulation of mating location and acceptance of new mating partners following mating to sexual satiety: The Coolidge effect demonstrated in the female golden hamster. Behav Neural Biol 36: 416--421, 1982. 17. Lisk, R. D., J. Zeiss and L. A. Ciaccio. The influence of olfaction on sexual behavior in the male golden hamster (Mesocricetus auratus). J Exp Zool 181: 69-78, 1972.

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